1. Field of the Invention
The present invention relates generally to antimicrobial agents and, more specifically, to crosslink-stabilized indolicidin analogs and methods of using the analogs to reduce-or inhibit microbial growth or survival.
2. Background Information
Infections by microorganisms, including bacteria, viruses and fungi, are a major cause of human morbidity and mortality. Although anyone can be a victim of such infection, the sick and elderly are particularly susceptible. For example, hospitalized patients frequently acquire secondary infections due to a combination of their weakened condition and the prevalence of microorganisms in a hospital setting. Such opportunistic infections result in increased suffering of the patient, increased length of hospitalization and, consequently, increased costs to the patient and the health care system. Similarly, the elderly, particularly those living in nursing homes or retirement communities, are susceptible to infections because of their close living arrangement and the impaired responsiveness of their immune systems.
Numerous drugs are available for treating infections by certain microorganisms. In particular, various bacterial infections have been amenable to treatment by antibiotics. However, the prolonged use of antibiotics since their discovery has resulted in the selection of bacteria that are relatively resistant to these drugs. Furthermore, few if any drugs are effective against microorganisms such as viruses. As a result, continuing efforts are being made to identify new and effective agents for treating infections by a variety of microorganisms.
The identification of naturally occurring compounds that act as antimicrobial agents has provided novel and effective drugs. Many organisms protect themselves by producing natural products that are toxic to other organisms. Frogs, for example, produce a class of peptides, magainins, that are highly toxic if ingested, thus providing a defense mechanism for the frog against potential predators. Magainins have been purified and shown to have antimicrobial activity, thus providing a natural product useful for reducing or inhibiting microbial infections.
Natural products useful as antimicrobial agents also have been purified from mammalian organisms, including humans. For example, the defensins are a class of peptides that have been purified from mammalian neutrophils and demonstrated to have antimicrobial activity. Similarly, indolicidin is a peptide that has been isolated from bovine neutrophils and has antimicrobial activity, including activity against viruses, bacteria, fungi and protozoan parasites. Thus, naturally occurring compounds provide a source of drugs that are potentially useful for treating microbial infections.
Upon identifying naturally occurring peptides useful as antimicrobial agents, efforts began to chemically modify the peptides to obtain analogs having improved properties. Such efforts have resulted, for example, in the identification of indolicidin analogs which, when administered to an individual, have increased selectivity against the infecting microorganisms as compared to the individual""s own cells. Thus, the availability of naturally occurring antimicrobial agents has provided new drugs for treating microbial infections and has provided a starting material to identify analogs of the naturally occurring molecule that have desirable characteristics.
Although such natural products and their analogs have provided new agents for treating microbial infections, it is well known that microorganisms can become resistant to drugs. Thus, a need exists to identify agents that effectively reduce or inhibit the growth or survival of microorganisms. The present invention satisfies this need and provides additional advantages.
The present invention relates to crosslink-stabilized analogs of indolicidin, which is a naturally occurring peptide having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (xe2x80x9cIndol 1-13;xe2x80x9d SEQ ID NO: 1). As disclosed herein, crosslink-stabilized indolicidin analogs (xe2x80x9cX-indolicidin analogsxe2x80x9d) of the invention are characterized, in part, by having an intrapeptide crosslink formed, for example, between two Trp residues, to form a di-tryptophan crosslink.
An X-indolicidin analog has the structure: X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8 wherein X1 is Ile, Leu, Val, Ala, Gly or absent; X2 is Ile, Leu, Val, Ala, Gly or absent; X3 is Pro or absent; X4 is Trp, Phe, Cys, Glu, Asp, Lys, AlaL or absent; X5 is Arg, Lys or absent; X6 is Trp, Phe, Cys, Glu, Asp, Lys or AlaL; X7 is Arg, Lys or absent; and X8 is homoserine, Met, Met-X9-Met or absent, wherein X9 is at least one amino acid; provided that the analog contains at least two amino acid residues that are capable of forming a crosslink; and further provided that if X2 is absent, X1 is absent; if X3 is absent, X1 and X2 are absent; if X4 is absent, X1, X2 and X3 are absent; and if X5 is absent, X1, X2, X3 and X4 are absent. X-indolidicin analogs of the invention are exemplified by the peptide Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 3), where the underlining indicates a di-tryptophan crosslink formed between the first and last underlined Trp residues; and by the peptide Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4), where underlining indicates a disulfide crosslink formed between the first and last underlined Cys residues. X-indolicidin analogs have broad spectrum antimicrobial activity.
The invention also provides fusion polypeptides comprising an X-indolicidin analog and a peptide of interest, which can be useful, for example, for facilitating purification of an expressed indolicidin analog. In addition, the invention provides nucleic acid molecules encoding X-indolicidin analogs of the invention, for example, disulfide crosslinked analogs, as well as precursors of such analogs and fusion polypeptides comprising such analogs.
The invention also relates to methods of using an X-indolicidin analog to reduce or inhibit microbial growth or survival in an environment capable of sustaining microbial growth or survival by contacting the environment with the X-indolicidin analog. As such, the invention provides methods of reducing or inhibiting microbial growth or survival on a solid surface, for example, surgical instruments, hospital surfaces, and the like. In addition, methods of the invention are useful for reducing or inhibiting microbial growth or survival in an individual, particularly a mammal such as a human. Thus, the invention provides methods of treating an individual suffering from a pathology caused, at least in part, by microbial infection, by administering an X-indolicidin analog to the individual, thereby reducing the severity of the pathologic condition.
FIG. 1 shows the nucleotide sequence encoding poly-(Indol(1-13)-Met-Ala-Arg-Ile-Ala-Met)3, (SEQ ID NO: 2) which encodes three copies of Indol 1-13, each separated by Met-Ala-Arg-Ile-Ala-met (SEQ ID NO: 2). The coding (sense) strand is shown in capital letters (SEQ ID NO:11), the antisense strand is shown in lower case letters (SEQ ID NO: 13), and the encoded amino acid sequence (SEQ ID NO:12) is shown using the three letter code (xe2x80x9cStpxe2x80x9d indicates STOP codon). The nucleotide and amino acid sequences correspond to SEQ ID NOS: 11 and 12, respectively. Eco RI and Sal I restriction endonuclease sites are indicated. The enterokinase recognition site is singly underlined, with the double arrow indicating the cleavage site. The single arrows denote cyanogen bromide cleavage sites. Dotted underlined tetranucleotide sequences correspond to overlaps in oligonucleotides used for ligation. Double underlined sequences denote primers used for PCR amplification (see Example I.C).
FIG. 2 shows the dose dependent microbistatic activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of Escherichia coli ML35.
FIG. 3 shows the dose dependent microbistatic activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of Cryptococcus neoformans 271A.
FIG. 4 shows the dose dependent microbistatic activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of Staphylococcus aureus 207A.
FIG. 5 shows the dose dependent microbistatic activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3) (inverted triangles) on growth of Candida albicans 16820.
FIG. 6 shows the dose dependent microbicidal activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of E. coli ML35.
FIG. 7 shows the dose dependent microbicidal activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of C. neoformans 271A.
FIG. 8 shows the dose dependent microbicidal activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3)(inverted triangles) on growth of S. aureus 207A.
FIG. 9 shows the dose dependent microbicidal activity of indolicidin (Indol 1-13; SEQ ID NO:1)(closed circles) and X-indolicidin (Indol 1-13(W6,9); SEQ ID NO:3) (inverted triangles) on growth of C. albicans ML35.